The present invention relates generally to the field of peptides and other small molecules (i.e. peptide mimetics) as pharmaceutical and/or therapeutic agents, and to methods for identification and design of peptides and peptide mimetics having desired functional activities. Specifically, peptides and other small molecules derived from regions of interacting intracellular signaling proteins are provided. More specifically, peptides and other small molecules derived from regions of the Gxcex2 subunit of heterotrimeric GTP binding proteins are provided. Such molecules include specific agonists and antagonists of Gxcex2 downstream effectors, including adenylyl cyclase and phospholipase C. Such molecules are targeted to predicted regions of interaction between intracellular signaling proteins and tested for activity in functional assays using methods of the invention. One major advantage of the invention is the incorporation of three-dimensional structural information in models used for predicting interaction surfaces between intracellular proteins. Another major advantage is the ability to distinguish, within a predicted interaction surface, a signal transfer region from a general binding domain. Resolution of such signal transfer regions from general binding domains is useful for prediction and validation of pharmacologic and therapeutic agonists and antagonists.
The ability to target a desired drug intervention to a specific site in a biological system underlies the rational design of safe and effective drugs. Past drug design efforts have often focused on development of molecules believed to interact with cell surface receptors. For example, high-throughput assays have been used to screen synthetic organic compounds to identify molecules interacting with an extracellular domain of a cell surface receptor (Tian et al., 1998, A small, nonpeptidyl mimic of granulocyte-colony-stimulating factor, Science 281, 257-259). Further, methods have been developed for determining whether a candidate compound is an agonist of a peptide hormone receptor (see Kopin et al., U.S. Pat. No. 5,750,353, issued May 12, 1998, Assay for non-peptide agonists to peptide hormone receptors). Peptides and mimetics have also been developed based on the transmembrane domains of G-protein-coupled receptors (Bouvier et al., Jan. 8, 1998, Peptides and peptidomimetic compounds affecting the activity of G-protein-coupled receptors by altering receptor oligomerization, International Publication No. WO 98/00538). Examples of other extracellular ligands for which peptide mimetics have been developed include erythropoietin and TNFxcex1 (Wrighton et al., 1997, Increased potency of an erythropoietin peptide mimetic through covalent dimerization, Nature Biotechnology 15, 1261-1265; Takasaki et al., 1997, Structure-based design and characterization of exocyclic peptidomimetics that inhibit TNFxcex1 binding to its receptor, Nature Biotechnology 15, 1266-1270). Finally, distinct regions of peptide hormones have even been considered for design of receptor antagonists (Portoghese et al., 1990, Design of peptidomimetic xcex4 opioid receptor antagonists using the message-address concept, J. Med. Chem. 33, 1714-1720).
Heterotrimeric GTP-binding proteins (G proteins) consisting of Gxcex1xcex2xcex3 subunits are ubiquitous signal transduction proteins that play essential roles in intracellular communication (see e.g. DeVivo and Iyengar, 1994, G protein pathways: signal processing by effectors, Molec. Cell. Endocrinol. 100, 65-70). For example, the enzymatic production of cyclic AMP (cAMP) via adenylyl cyclases is regulated by G proteins (Smit and Iyengar, 1998, Mammalian adenylyl cyclases, Adv. Sec. Mess. Phosphoprot. Res. 32, 1-21; Iyengar, 1993, Multiple families of Gs-regulated adenylyl cyclases, Adv. Sec. Mess. Phosphoprot. Res. 28, 27-36; Pieroni et al., 1993, Signal recognition and integration by Gs-stimulated adenylyl cyclases, Curr. Opin. Neurobiol. 3, 345-351; Weng et al., 1996, G beta subunit interacts with a peptide encoding region 956-982 of adenylyl cyclase 2, cross-linking of the peptide to free G beta gamma but not the heterotrimer, J. Biol. Chem. 271, 26445-264488; Harry et al., 1997, Differential regulation of adenylyl cyclases by G alphas, J. Biol. Chem. 272, 19017-19021). G proteins provide a versatile system for investigation of intracellular protein-protein interactions by virtue of their interactions with multiple downstream effectors. For example, G protein xcex2xcex3 subunits regulate the activity of not on adenylyl cyclase but also phospholipase C-xcex22, calcium channels, potassium channels, and xcex2-adrenergic receptor kinase (see e.g. Ford et al., 1998, Molecular basis for interactions of G protein xcex2xcex3 subunits with effectors, Science 280, 1271-1274).
Drug intervention beyond the cell surface, i.e. at intracellular protein-protein interaction sites, would broaden the array of potential targets for achieving a desired therapeutic effect. Intracellular targets may also provide intervention points having enhanced specificity compared to drugs targeted strictly at cell surface receptors. The ability to use intracellular interacting proteins as therapeutic targets for drug design has been less clearly established, however. One reason may be that an intracellular protein-protein interaction, unlike a typical cell surface hormone-receptor interaction, will often involve a multiplicity of proteins. Thus, resolution of specific interactions among three or more proteins will often be necessary to carry out design of safe and effective drugs. Accordingly, a need exists for a generally-applicable approach for identification of peptides and mimetics thereof having selective activity at a chosen intracellular site of action.
This invention provides peptides and other small molecules derived from regions of intracellular interacting proteins and methods for identification of such molecules. More specifically, the present invention provides peptides and other small molecules derived from regions of Gxcex2 proteins which function as agonists or antagonists of adenylyl cyclase or phospholipase C-xcex22. The invention is based, at least in part, on the discovery of the inventors that it is possible to resolve, within a given intracellular signal transduction protein, a signal transfer region from a general binding domain. Such resolution provides a rational basis for design of agonists and antagonists of virtually any desired intracellular protein-protein interaction. The drug design methods of the invention utilize three-dimensional structural information for prediction of protein-protein interactions followed by evaluation of predictions in functional assays.
The present invention relates generally to the field of peptides and peptide mimetics as pharmaceutical and/or therapeutic agents. More particularly, the present invention relates to peptides and other small molecules (e.g. peptide mimetics) derived from regions of Gxcex2 proteins and their use as pharmaceutical and/or therapeutic agents. For example, peptides and derivatives thereof for modulating adenylyl cyclase and phospholipase C-xcex22 activities are provided. Still further, methods for identification of peptides and derivatives thereof useful for modulating a chosen effector-of-interest among various effectors are provided. One advantage of the methods of the invention is the use of structural modeling information to predict and validate pharmacologic and therapeutic agents.
Predictions about effector interactions of Gxcex2 proteins have been made using a combination of molecular modeling and experimental validation in which the predictions of the model are tested. Through an iterative process involving cycles of structural modeling followed by experimental testing, precise definition of individual effector domains within a Gxcex2 signaling protein has been achieved. This validated procedure has general applicability for drug design targeted at other intracellular protein-protein interactions in virtually any intracellular signal transduction pathway.
This invention provides an isolated Gxcex2 peptide or derivative thereof. This invention provides a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. In one embodiment, a derivative of a peptide is capable of immunospecific binding to an antipeptide antibody. In a preferred embodiment, a peptide or a derivative thereof displays only one functional activity of an intracellular signaling protein from which it is derived. This invention provides a purified fragment of a peptide, which fragment displays one or more functional activities of an intracellular signaling protein. This invention provides a purified fragment of a peptide comprising a region of the peptide selected from the group consisting of an adenylyl cyclase interaction region and a phospholipase C interaction region. This invention provides a purified molecule comprising the fragment. This invention provides a chimeric peptide comprising the fragment, which fragment consists of at least 6 amino acids fused by a covalent bond to an amino acid sequence of a second peptide.
This invention provides a purified antibody or an antigen-binding derivative thereof capable of immunospecific binding to a peptide selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 and not to a protein from which the peptide was derived. In one embodiment, the antibody is polyclonal. In another embodiment, the antibody is monoclonal.
This invention provides a method of making a recombinant protein comprising: (a) growing a recombinant cell containing a nucleic acid comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10 such that the recombinant protein is expressed by the cell; and (b) recovering the expressed recombinant protein. Further, this invention provides a purified recombinant protein produced by said method. Any method known in the art may be used for growing the recombinant cell (see e.g. Freshney, 1994, Culture of animal cells, A manual of basic technique, 3d ed., Wiley-Liss, Inc., New York). Any method known in the art may be used for recovering the recombinant protein, such as routine size exclusion chromatography, molecular tagging with histidine and purification on a nickel column, etc.
This invention provides a pharmaceutical composition comprising: (a) a peptide or derivative thereof selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10; and (b) a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any carrier known to one skilled in the art.
This invention provides a method of identifying a peptide or derivative thereof having a biological activity of interest comprising: (a) providing a molecular model of an intracellular protein-protein interaction, which model predicts one or more interaction surfaces among a plurality of interacting proteins from three-dimensional structure information; and (b) testing a candidate interaction surface predicted by the molecular model by determining whether a peptide encoding at least a portion of the surface has a functional activity in a functional assay. In one embodiment, the functional activity is an agonist activity. In another embodiment, the functional activity is an antagonist activity.
This invention provides a method of identifying a functional activity of a Gxcex2 peptide comprising: (a) expressing a protein comprising a peptide selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10 in a biological system; and (b) measuring an effect of expression in a biological assay. In one embodiment, the biological system is selected from the group consisting of an animal cell culture and an experimental animal. In another embodiment, the experimental animal is selected from the group consisting of a fly (e.g. D. melanogaster), a worm (e.g. C. elegans), a fish (e.g. zebrafish), a rat, a mouse and a guinea pig. In yet another embodiment, the biological assay is selected from the group consisting of an adenylyl cyclase assay, a phospholipase C assay, a potassium channel assay, a calcium channel assay and a xcex2-adrenergic receptor kinase assay.
This invention provides a method of detecting an effect of expression of a recombinant protein comprising a peptide selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:10 on a signal transduction pathway, the method comprising: (a) expressing the recombinant protein in a cell culture or experimental animal already having a mutation in the signal transduction pathway; and (b) detecting the effect of expression in a biological assay. In one embodiment, the biological assay is selected from the group consisting of an adenylyl cyclase assay, a phospholipase Cxcex2 assay, a potassium channel assay and a calcium channel assay. In another embodiment, the mutation in the signal transduction pathway is in a gene selected from the group consisting of an adenylyl cyclase gene, a phospholipase C gene, a potassium channel gene and a calcium channel gene.
This invention provides a method of identifying a molecule that specifically binds to a peptide or derivative thereof selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, the method comprising: (a) contacting the peptide or derivative thereof with a plurality of molecules under conditions conducive to binding; and (b) identifying a molecule from the plurality of molecules that specifically binds to the peptide or derivative thereof.
The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.
FIGS. 1A and 1B. Regions of Gxcex2 involved in contacts with the AC2 956-982 peptide. (FIG. 1A) Ribbon diagram of the Gxcex2 backbone from the crystal structure of Gxcex2xcex3 (Sondek et al., 1996, Nature 379, 369-374; Lambright et al., 1996, Nature 379, 311-319); the residues in contact with the AC2 peptide are shown in pink (Weng et al., 1996, J. Biol. Chem. 271, 26445-26448). (FIG. 1B) Predicted core contacts between the AC2 956-982 peptide and Gxcex2. The AC2 peptide residues are in the blue boxes. The AC2 peptide residues are numbered 1-27 from the N terminus. Gxcex21 residues are in green boxes. The Gxcex21 residues are shown in the spatial sequence in which they are predicted to interact with the AC2 peptide.
FIGS. 2A-2E. Effects of the Gxcex286-105 peptides on AC2 and AC1 activities. (FIG. 2A) Ribbon diagram of the Gxcex2 backbone with residues 86-105 in yellow. Other residues in contact with the AC2 peptide are shown in pink. (FIG. 2B) Effect of the Gxcex286-105 peptide (TTN) and the M101N Gxcex286-105 mutant peptide (m-TTN) on basal, xcex1s* (2 nM), and various concentrations of TTN peptide on Gxcex2xcex3-stimulated AC2 activity in the presence of xcex1s* (2 nM) plus Gxcex2xcex3 (50 nM) stimulated AC2 activities. (FIG. 2C) Effect of various concentrations of TTN peptide on Gxcex2xcex3-stimulated AC2 activity in the presence of xcex1s* (2 nM). (FIG. 2D) Effect of TTN and m-TTN peptides on basal and CaM (100 nM) plus Gxcex2xcex3 (30 nM) regulated AC1 activities. (FIG. 2E) Effect of TTN and m-TTN peptides on basal and CaM (100 nM) stimulated AC1 activities.
FIGS. 3A-3C. Effects of the Gxcex2115-135 peptide on AC2 and AC1 activities. (FIG. 3A) Ribbon diagram of the Gxcex21 backbone with residues 115-135 in yellow. Other residues in contact with the AC2 peptide are shown in pink. (FIG. 3B) Effect of the Gxcex2115-135 peptide (GGL) and the Y124V Gxcex2115-135 mutant peptide (m-GGL) on basal, xcex1s* (2 nM), and xcex1s* (2 NM) plus Gxcex2xcex3 (50 nM) stimulated AC2 activities. (FIG. 3C) Effect of GGL and m-GGL peptides on basal, CaM (100 nM), or CaM (100 nM) plus Gxcex2xcex3 (30 nM) regulated AC1 activities.
FIG. 4. Schematic representation of the regions of Gxcex2 involved in interactions with Gxcex1 (outlined in green) and some regions that may interact with adenylyl cyclases 1 and 2 (outlined in red). The space-filling model of Gxcex2 was obtained from the crystallographic coordinates; Gxcex1 contact regions are those identified by Sigler and coworkers (Sondek et al., 1996, Nature 379, 369-374; Lambright et al., 1996, Nature 379, 311-319) from the crystal structure of the heterotrimer. The AC2 peptide interaction region was deduced from molecular modeling studies (Weng et al., 1996, J. Biol. Chem. 271, 26445-26448) and the functional data in FIG. 2 and FIG. 3 indicate that these regions may be involved in interactions with AC1 and AC2.
FIGS. 5A-C. Effects of varying concentrations of Gxcex286-105 peptide on PLC-xcex22 activity. FIG. 5A: Effects of Gxcex286-105 peptide on basal and Gxcex2xcex3 (100 nM) stimulated PLC-xcex22 activity. FIGS. 5B-C: Effects of Gxcex286-105 peptide and M101N Gxcex286-105 peptide on PLC-xcex22 activity.
FIGS. 6A-G. Effects of varying concentrations of Gxcex286-105 peptide and (FIGS. 6A-C) K89A, H91A, and R96A substituted peptides on PLC-xcex22 activity (FIGS. 6D-E) K89A, H91A, and R96A triple substituted peptide on basal (FIG. 6D) and Gxcex2xcex3 (100 nM) (FIG. 6E) stimulated PLC-xcex22 activity. (FIG. 6F) Effects of varying concentrations of Gxcex286-105 peptide and FLLT peptide on PLC-xcex22 activity. (FIG. 6G) Effects of 100 nM Gxcex2xcex3 and varying concentrations of Gxcex286-105 peptide on PLC-xcex22 and PLCXxcex2 activity.
FIGS. 7A-C. Effects of varying concentrations of Gxcex286-105 peptide and (FIG. 7A) S98A Gxcex286-105 peptide, (FIG. 7B) S97,98R Gxcex286-105 peptide, and (FIG. 7C) S97, 98D and S97, 98C peptides on PLC-xcex22 activity.
FIGS. 8A-8E. Effects of shorter peptides from Gxcex286-105 region on PLC-xcex22 activity. (FIG. 8A) Effects of 100 xcexcM Gxcex296-98, Gxcex296-101, and Gxcex289-101 peptides on PLC-xcex22 activity. (FIGS. 8B-C) Effects of varying concentrations of Gxcex296-101 peptide and S97, 98R (FIG. 8B) and S97, 98D (FIG. 8C) Gxcex296-101 peptides on PLC-xcex22 basal activity. Values for (FIG. 8A) are given as meanxc2x1SEM of three experiments.
FIGS. 9A-B. Effects of Gxcex2115-135 peptide on PLC-xcex22 activity. (FIG. 9A) Effects of 30 nM Gxcex2115-135 peptide and Y124V Gxcex2115-135 peptide on basal and Gxcex2xcex3 (100 nM) stimulated PLC-xcex22 activity. (FIG. 9B) Effect of varying concentrations of Gxcex2115-135 peptide on Gxcex2xcex3 (100 nM) stimulated PLC-xcex22 activity. Values for (FIG. 9A) are given as meanxc2x1SEM of three experiments.
FIG. 10. Ribbon diagram of Gxcex2xcex3.